Biology subjects

Kelliher, J. L.

Publications and source records attributed to Kelliher, J. L..

3 recordsLinked to original sources

bioRxiv2026

Manganese scavenging is required for virulence of the cytosolic pathogen Listeria monocytogenes

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

microbiology↗

bioRxiv2023

GpsB control of PASTA kinase activity in Listeria monocytogenes influences peptidoglycan synthesis during cell wall stress and cytosolic survival

The ability to respond quickly to changing environmental conditions in the host is critical for bacterial pathogens. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are a conserved family of kinases important for cell envelope stress responses in Firmicutes and Actinobacteria, including the cytosolic pathogen Listeria monocytogenes. As serine/threonine kinases, PASTA kinases phosphorylate multiple substrates, yet the mechanisms through which these substrates promote resistance to cell wall stress remain poorly understood. We previously identified GpsB as a target of PrkA, the PASTA kinase in L. monocytogenes, through a phosphoproteomics screen. Here, we demonstrate that GpsB can be directly phosphorylated by PrkA, and that mutation of the PrkA-dependent phosphosite T88 to a phosphoablative residue enhances PrkA activity in vitro. We find that relative to a strain of L. monocytogenes harboring the gpsBT88A allele, a strain with the phosphomimetic gpsBT88D allele is more sensitive to the cephalosporin antibiotic ceftriaxone and has a diminished capacity to increase peptidoglycan synthesis during stress. We find that GpsB-dependent control of PrkA activity is required for optimal survival and replication of L. monocytogenes in the macrophage cytosol. Finally, we show that GpsB is required for full virulence of L. monocytogenes, due in part to its role in modulating PrkA activity. Cumulatively, these results demonstrate that phosphorylative feedback between GpsB and PrkA is important for the ability of L. monocytogenes to respond to cell wall stress, survive in its cytosolic niche, and cause infection.

microbiology↗

bioRxiv2021

PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine- associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a {beta}-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a {Delta}prkA mutant to nearly wild-type levels. While a {Delta}prkA mutant is defective for PG synthesis during cell wall stress, a double {Delta}reoM {Delta}prkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a {Delta}prkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence. AUTHOR SUMMARYMany antibiotics target bacterial cell wall biosynthesis, justifying continued study of this process and the ways bacteria respond to cell wall insults, especially in the context of infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are master regulators of cell wall stress responses in bacteria and are conserved in several major pathogens, including Listeria monocytogenes, Staphylococcus aureus, and Mycobacterium tuberculosis. We previously showed that the PASTA kinase in L. monocytogenes, PrkA, is essential for the response to cell wall stress and virulence. PrkA is also necessary for adaptation to the cytosol, which L. monocytogenes requires during its infectious lifecycle. In this work, we combined orthogonal phosphoproteomic and genetic screens to identify PrkA substrates in L. monocytogenes. We show that phosphoregulation of one candidate from both screens, ReoM, increases peptidoglycan synthesis and that this regulation is required for cytosolic survival and virulence. We also demonstrate that the PASTA kinase-ReoM pathway regulates cell wall stress responses in another significant pathogen, methicillin-resistant S. aureus. Additionally, we uncover a PrkA-independent role for ReoM in vivo in L. monocytogenes, suggesting a need for nuanced modulation of peptidoglycan synthesis during infection. Cumulatively, this study provides new insight into how bacterial pathogens control cell wall synthesis during infection.

microbiology↗