Person: Kieser, Karen
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Kieser
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Karen
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Kieser, Karen
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Publication Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance(John Wiley and Sons Inc., 2016) Filippova, Ekaterina V.; Kieser, Karen; Luan, Chi‐Hao; Wawrzak, Zdzislaw; Kiryukhina, Olga; Rubin, Eric; Anderson, Wayne F.Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. Database Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW.Publication Genomic Analysis Identifies Targets of Convergent Positive Selection in Drug Resistant Mycobacterium tuberculosis(2013) Farhat, Maha; Shapiro, B Jesse; Kieser, Karen; Sultana, Razvan; Jacobson, Karen R; Victor, Thomas C; Warren, Robin M; Streicher, Elizabeth M; Calver, Alistair; Sloutsky, Alex; Kaur, Devinder; Posey, Jamie E; Plikaytis, Bonnie; Oggioni, Marco R; Gardy, Jennifer L; Johnston, James C; Rodrigues, Mabel; Tang, Patrick K C; Kato-Maeda, Midori; Borowsky, Mark L; Muddukrishna, Bhavana; Kreiswirth, Barry N; Kurepina, Natalia; Galagan, James; Gagneux, Sebastien; Birren, Bruce; Rubin, Eric; Lander, Eric S; Sabeti, Pardis; Murray, MeganMycobacterium tuberculosis is successfully evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including the genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly and 7 previously sequenced M. tuberculosis genomes, we identified genomewide signatures of positive selection specific to the 47 resistant genomes. By searching for convergent evolution, the independent fixation of mutations at the same nucleotide site or gene, we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode pathways of cell wall biosynthesis, transcriptional regulation and DNA repair. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.Publication Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria(Public Library of Science, 2015) Kieser, Karen; Boutte, Cara; Kester, Jemila; Baer, Christina E.; Barczak, Amy; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah; Rubin, EricCell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress.Publication Spatiotemporal Control of Mycobacterium Tuberculosis Cell Wall Biogenesis by the Peptidoglycan Synthase PonA1(2015-05-04) Kieser, Karen; Goldberg, Marcia; Wirth, Dyann; Burleigh, Barbara; Dove, SimonMycobacterium tuberculosis causes one of the most pernicious infectious diseases of humankind – tuberculosis, which is still a global problem in the 21st century. Drug-resistant M. tuberculosis infections are on the rise, necessitating novel drug development. A particularly fruitful avenue for drug development is the bacterial cell wall, as it is a structure required for bacterial survival and is accessible to small molecules. This complex structure must be dynamically remodeled every cell cycle to promote bacterial growth, and our understanding of how M. tuberculosis governs this process and the enzymes involved is rudimentary. My work investigates how M. tuberculosis governs the synthesis and remodeling of an essential component of its cell wall – peptidoglycan. For cells to grow and divide, the peptidoglycan layer must be actively metabolized. Failures in the peptidoglycan structure result in cell lysis and death, which is why peptidoglycan synthesis is often targeted by antibiotics, such as penicillin. Hence, peptidoglycan must be static enough to resist interior turgor pressure and exterior insults (like antibiotics) and yet it must also be plastic enough to allow local restructuring to promote cell growth. How does the cell govern these paradoxical processes in both space and time to ensure survival? I used a key peptidoglycan synthase, PonA1, to understand how M. tuberculosis engineers its cell wall during growth and division. PonA1 localizes to the cell septum during division, where it interacts with the essential peptidoglycan hydrolase RipA and modulates RipA activity during cell separation. PonA1 also localizes to the cell pole where it promotes expansion of the peptidoglycan layer of the cell envelope. I found that PonA1 is critical for the proper spatial organization of new pole growth and that phosphorylation of PonA1 modulates the rate of cell elongation at the pole. In an effort to expand our understanding of how M. tuberculosis remodels its cell wall, I performed a whole genome mutagenesis screen to identify genetic partners of PonA1. I found factors critical for peptidoglycan synthesis, namely PonA2 and LdtB, a nonclassical transpeptidase that synthesizes the predominant crosslink in mycobacterial peptidoglycan. Further genetic and biochemical studies showed that simultaneous loss of PonA2 and LdtB is lethal to M. tuberculosis, suggesting an opportunity to develop chemotherapeutics targeting 3-3 transpeptidases like LdtB. These studies also suggested that enzymatic pathways that govern M. tuberculosis cell wall synthesis are spatially resolved. My work identified genetic networks that govern cell wall construction in M. tuberculosis and showed the critical role of the peptidoglycan synthase PonA1 in controlling growth at the cell pole. Defining the spatiotemporal dynamics of key cell wall synthase proteins is important for understanding M. tuberculosis’ physiology. My work expands our understanding of how a prevalent human pathogen governs the synthesis of its cell wall, a critical source for novel drug development.Publication Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria(Public Library of Science, 2013) Chao, Michael C.; Kieser, Karen; Minami, Shoko; Mavrici, Daniela; Aldridge, Bree; Fortune, Sarah; Alber, Tom; Rubin, EricPeptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.