Brianna Dalesandro

Biophys J. 2023 Feb 10;122(3S1):300a. doi: 10.1016/j.bpj.2022.11.1692.

NO ABSTRACT

PMID:36783503 | DOI:10.1016/j.bpj.2022.11.1692

Proc Natl Acad Sci U S A. 2023 Feb 21;120(8):e2215237120. doi: 10.1073/pnas.2215237120. Epub 2023 Feb 14.

ABSTRACT

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. β-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria.

PMID:36787358 | PMC:PMC9974482 | DOI:10.1073/pnas.2215237120

ACS Infect Dis. 2021 Feb 12;7(2):461-470. doi: 10.1021/acsinfecdis.0c00809. Epub 2021 Jan 20.

ABSTRACT

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, Mtb relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of β-lactam antibiotics to study two classes of enzymes that maintain Mtb's peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of β-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of Mtb. All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify β-lactam targets by mass spectrometry, including PBPs, LDTs, and the β-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that Mtb dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.

PMID:33470787 | PMC:PMC8096124 | DOI:10.1021/acsinfecdis.0c00809

Curr Opin Chem Biol. 2020 Aug;57:50-57. doi: 10.1016/j.cbpa.2020.04.006. Epub 2020 Jun 9.

ABSTRACT

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.

PMID:32531742 | DOI:10.1016/j.cbpa.2020.04.006

Proc Natl Acad Sci U S A. 2021 May 11;118(19):e2101989118. doi: 10.1073/pnas.2101989118.

ABSTRACT

The gram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun's lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG-Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly identified paralog of l,d-transpeptidases in E. coli, is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG-Lpp cross-links, suggesting a role for LdtF in the regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.

PMID:33941679 | PMC:PMC8126863 | DOI:10.1073/pnas.2101989118

Elife. 2022 Jun 9;11:e72863. doi: 10.7554/eLife.72863.

ABSTRACT

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

PMID:35678393 | PMC:PMC9249393 | DOI:10.7554/eLife.72863

ACS Infect Dis. 2022 Sep 9;8(9):1948-1961. doi: 10.1021/acsinfecdis.2c00321. Epub 2022 Aug 16.

ABSTRACT

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of Ldt_Ab from the clinically important pathogen, Acinetobacter baumannii. We show that A. baumannii survives inactivation of Ldt_Ab alone or in combination with PBP1b or PBP2, while simultaneous inactivation of Ldt_Ab and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 β-lactam antibiotics tested decreased 2- to 8-fold for the Ldt_Ab deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the Ldt_Ab + PBP2 deletion mutant. Mass spectrometry experiments showed that Ldt_Ab forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that Ldt_Ab has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

PMID:35973205 | PMC:PMC9764404 | DOI:10.1021/acsinfecdis.2c00321

J Bacteriol. 2023 Jan 26;205(1):e0042422. doi: 10.1128/jb.00424-22. Epub 2022 Dec 21.

ABSTRACT

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most β-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

PMID:36541811 | PMC:PMC9879121 | DOI:10.1128/jb.00424-22

Nat Commun. 2022 Dec 27;13(1):7962. doi: 10.1038/s41467-022-35528-3.

ABSTRACT

The D,D-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of β-lactam antibiotics that block peptidoglycan polymerization. β-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a β-lactam-insensitive L,D-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by β-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond L,D-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of β-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of β-lactams involves self-promoted penetration through the outer membrane.

PMID:36575173 | PMC:PMC9794725 | DOI:10.1038/s41467-022-35528-3

Microb Genom. 2022 Dec;8(12):mgen000902. doi: 10.1099/mgen.0.000902.

ABSTRACT

Staphylococcus aureus is a leading cause of skin and soft tissue infections and systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with β-linked GlcNAc at the C-4 (β1,4-GlcNAc) and the C-3 position (β1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally present within the S. aureus population, whereas a smaller proportion co-contain either tarM or tarP. To gain more insight into the presence and genetic variation of tarS, tarM and tarP in the S. aureus population, we analysed a collection of 25 652 S. aureus genomes within the PubMLST database. Over 99 % of isolates contained tarS. Co-presence of tarS/tarM or tarS/tarP occurred in 37 and 7 % of isolates, respectively, and was associated with specific S. aureus clonal complexes. We also identified 26 isolates (0.1 %) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

PMID:36748528 | PMC:PMC9837562 | DOI:10.1099/mgen.0.000902

mBio. 2023 Feb 28;14(1):e0008923. doi: 10.1128/mbio.00089-23. Epub 2023 Feb 8.

ABSTRACT

Most bacteria have a peptidoglycan cell wall that determines their cell shape and helps them resist osmotic lysis. Peptidoglycan synthesis depends on the translocation of the lipid-linked precursor lipid II across the cytoplasmic membrane by the MurJ flippase. Structure-function analyses of MurJ from Thermosipho africanus (MurJ_Ta) and Escherichia coli (MurJ_Ec) have revealed that MurJ adopts multiple conformations and utilizes an alternating-access mechanism to flip lipid II. MurJ_Ec activity relies on membrane potential, but the specific counterion has not been identified. Crystal structures of MurJ_Ta revealed a chloride ion bound to the N-lobe of the flippase and a sodium ion in its C-lobe, but the role of these ions in transport is unknown. Here, we investigated the effect of various ions on the function of MurJ_Ta and MurJ_Ec in vivo. We found that chloride, and not sodium, ions are necessary for MurJ_Ta function, but neither ion is required for MurJ_Ec function. We also showed that murJ_Ta alleles encoding changes at the crystallographically identified sodium-binding site still complement the loss of native murJ_Ec, although they decreased protein stability and/or function. Based on our data and previous work, we propose that chloride ions are necessary for the conformational change that resets MurJ_Ta after lipid II translocation and suggest that MurJ orthologs may function similarly but differ in their requirements for counterions. IMPORTANCE The biosynthetic pathway of the peptidoglycan cell wall is one of the most favorable targets for antibiotic development. Lipid II, the lipid-linked PG precursor, is made in the inner leaflet of the cytoplasmic membrane and then transported by the MurJ flippase so that it can be used to build the peptidoglycan cell wall. MurJ functions using an alternating-access mechanism thought to depend on a yet-to-be-identified counterion. This study fills a gap in our understanding of MurJ's energy-coupling mechanism by showing that chloride ions are required for MurJ in some, but not all, organisms. Based on our data and prior studies, we propose that, while the general transport mechanism of MurJ may be conserved, its specific mechanistic details may differ across bacteria, as is common in transporters. These findings are important to understand MurJ function and its development as an antibiotic target.

PMID:36752629 | PMC:PMC9973255 | DOI:10.1128/mbio.00089-23

Angew Chem Int Ed Engl. 2023 Jan 26. doi: 10.1002/anie.202217777. Online ahead of print.

ABSTRACT

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

PMID:36700874 | DOI:10.1002/anie.202217777

ACS Omega. 2023 Jan 17;8(4):4026-4037. doi: 10.1021/acsomega.2c06844. eCollection 2023 Jan 31.

ABSTRACT

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.

PMID:36743041 | PMC:PMC9893251 | DOI:10.1021/acsomega.2c06844

Molecules. 2023 Jan 10;28(2):690. doi: 10.3390/molecules28020690.

ABSTRACT

Posttranslational modifications (PTMs) play an important role in cell signaling and they are often deregulated in disease. This review addresses recent advances in the development of heterobifunctional small molecules that enable targeting or hijacking PTMs. This emerging field is spearheaded by proteolysis-targeting chimeras (PROTACs), that induce ubiquitination of their targets and, thus, tag them for degradation by the proteasome. Within the last decade, several improvements have been made to enhance spatiotemporal control of PROTAC-induced degradation as well as cell permeability. Inspired by the success story of PROTACs, additional concepts based on chimeric small molecules have emerged such as phosphatase-recruiting chimeras (PhoRCs). Herein, an overview of strategies causing (de-)phosphorylation, deubiquitination as well as acetylation is provided, and the opportunities and challenges of heterobifunctional molecules for drug discovery are highlighted. Although significant progress has been achieved, a plethora of PTMs have not yet been covered and PTM-inducing chimeras will be helpful tools for chemical biology and could even find application in pharmacotherapy.

PMID:36677746 | PMC:PMC9867477 | DOI:10.3390/molecules28020690

EBioMedicine. 2023 Feb;88:104439. doi: 10.1016/j.ebiom.2023.104439. Epub 2023 Jan 27.

ABSTRACT

BACKGROUND: Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis.

METHODS: Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization.

RESULTS: We selected for further study the conserved surface polysaccharide Poly-β-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen.

INTERPRETATION: Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections.

FUNDINGS: ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.

PMID:36709579 | PMC:PMC9900374 | DOI:10.1016/j.ebiom.2023.104439