Biology subjects

Boutte, C. C.

Publications and source records attributed to Boutte, C. C..

9 recordsLinked to original sources

bioRxiv2026

A c-di-AMP-controlled glutamine synthesis pathway promotes persistence of Staphylococcus aureus thymidine-dependent small colony variants in the lung

Children with cystic fibrosis (CF) commonly harbor Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs), which are associated with reduced lung function and increased respiratory exacerbations. How TD-SCVs survive in the thymidine-limited CF lung is unknown. Here, we show that TD-SCVs exhibit impaired glutamine uptake and depend on c-di-AMP-regulated de novo glutamine synthesis for survival in the murine lung. We found that transcription of the glutamine synthetase gene glnA is cooperatively repressed by the transcriptional regulator GlnR, the c-di-AMP-binding protein PstA, and GlnA itself. Glutamine starvation elevates c-di-AMP levels, relieving repression by this ternary complex and promoting glutamine synthesis. Reducing c-di-AMP levels causes a profound growth defect in TD-SCVs under low-thymidine conditions, which is rescued by glnA overexpression. Moreover, pharmacological inhibition of GlnA markedly impairs TD-SCV growth in murine lung. These findings elucidate the molecular mechanism underlying S. aureus TD-SCV survival during infection and identify glutamine synthesis as a promising therapeutic target for treating infections caused by antifolate-resistant bacteria.

microbiology↗

bioRxiv2026

Polar growth factor PgfA regulates polar peptidoglycan synthesis as well as mycolate synthesis in Mycobacterium smegmatis

The mycobacterial cell envelope consists of multiple covalently linked layers that must be synthesized in a coordinated manner to maintain cell wall integrity. Despite the importance of this coordination, its molecular mechanisms remain poorly understood. PgfA (polar growth factor A) interacts with trehalose monomycolate lipids (TMMs) (1) and the TMM transporter MmpL3 (1, 2). PgfA promotes TMM transport in the periplasm and functions as an upstream regulator of polar growth. How TMM transport is linked to the expansion of the entire multi-layered cell wall is unclear. Here, we provide evidence that PgfA regulates peptidoglycan metabolism. We show that PgfA localization correlates with peptidoglycan metabolism and that PgfA can function as both an activator and inhibitor of peptidoglycan metabolism. We further explore the role of TMMs in polar growth and find evidence that periplasmic TMMs are a signaling molecule that may regulate polar peptidoglycan metabolism. Finally, we find an epistatic connection between PgfA overexpression and altered TMM levels that suggests that PgfA and TMMs work in the same pathway to regulate peptidoglycan metabolism. Our data are consistent with a model in which TMM-free PgfA inhibits peptidoglycan metabolism, while TMM-bound PgfA promotes polar peptidoglycan metabolism. This work identifies PgfA as a key protein that coordinates synthesis of the peptidoglycan and mycolic acid envelope layers. ImportanceThe mycobacterial cell envelope consists of multiple covalently linked layers whose synthesis must be coordinated to maintain cell integrity. Despite decades of research on individual envelope components, the molecular mechanisms coordinating synthesis of different layers remain poorly understood. Here, we identify PgfA as a key regulatory protein that coordinates peptidoglycan and mycolate synthesis in mycobacteria. PgfA has both inhibitory and stimulatory effects on peptidoglycan metabolism, depending on the context. Our findings suggest PgfA may act as a regulator that senses mycolate precursor availability and prevents envelope imbalance when these precursors are limiting. This work provides new insight into how mycobacteria coordinate the synthesis of their complex cell envelope, with implications for better understanding mycobacterial physiology and developing antimycobacterial therapeutics.

microbiology↗

bioRxiv2026

SepIVA has a function in activating the mycobacterial divisome, which is inhibited during DNA damage

Bacterial cell division is a tightly regulated process that is carried out by a complex of proteins called the divisome, which is assembled in a defined sequential order. Upon assembly the complex is allosterically activated, which stimulates cell wall synthesis at the division site. Bacteria inhibit division during DNA damage by blocking either divisome assembly or activation. While the regulation of cell division is known to be important during M. tuberculosis infection, little is known about mycobacterial mechanisms of divisome function and regulation in DNA damage. By using M. smegmatis as a model organism, we find here that divisome factor SepIVA is involved in septum initiation, and that it is recruited to the mid-cell by FtsQ but it is not a recruitment factor itself. We find that a sepIVA loss-of-function defect can be suppressed by overexpression of ftsW, supporting a role for SepIVA in activation of the divisome complex. When cell division is inhibited during DNA damage, we find that SepIVA is delocalized from the division site, while the septal localization of FtsZ, FtsQ and FtsW are not impacted. We also find that the interaction between FtsQ and SepIVA is inhibited during DNA damage. Our results suggest that SepIVA is a trigger factor for activation of cell division during normal growth, and show that the signaling to inhibit cell division during DNA damage involves inhibition of its interaction with FtsQ. IMPORTANCECell division is critical for bacterial cells to propagate and cause infection. Despite its importance, division is a dangerous process as it requires building and subsequently hydrolyzing new cell wall material, in a place where the chromosome typically resides. If cell division is done before chromosome segregation is completed, or if cell wall metabolism is improperly regulated, then cell death results. The divisome is a protein complex that regulates cell division - coordinating it with the status of the chromosome and ensuring that the cell wall metabolic enzymes are carefully controlled. Many of the proteins in the divisome complex are highly conserved across bacterial Phyla; however, the factor that stimulates the complex to activate and initiate cell wall synthesis is not widely conserved. Here, we study the activation of the divisome in Mycobacterium smegmatis, a model for cell physiology of Mycobacterium tuberculosis. We find evidence that SepIVA, a protein found only in Actinobacteria, is likely the factor that stimulates activation of the divisome in mycobacteria. We also show that the association of SepIVA with the divisome is blocked under DNA damage, when cell division is inhibited. These results provide a model for the regulation of cell division in mycobacteria in growth and stress, and also provide insights into how bacteria with different types of septa may regulate division differently.

microbiology↗

bioRxiv2024

MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3s polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.

microbiology↗

bioRxiv2022

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum

microbiology↗

bioRxiv2022

Polar growth protein Wag31 undergoes changes in homo-oligomeric network topology, and has distinct functions at both cell poles and the septum

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. Wag31 homo-oligomerizes in a network at the poles, but it is not known how the structure of this network affects Wag31 function. In this study we used a protein fragment complementation assay to identify Wag31 residues involved in homo-oligomeric interactions, and found that amino acids all along the length of the protein mediate these interactions. We then used both N-terminal and C-terminal splitGFP fusions to probe Wag31 network topology at different sites in the cell, and found that Wag31 C-terminal-C-terminal interactions predominate at the septa, while C-terminal-C-terminal and C-terminal-N-terminal interactions are found equally at the poles. This suggests the Wag31 network is formed through an ordered series of associations. We then dissected Wag31s functional roles by phenotyping a series of wag31 alanine mutants; these data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. This work establishes new functions for Wag31, and indicates that changes in Wag31 homo-oligomeric network topology may contribute to cell wall regulation in mycobacteria. ImportanceMany bacteria restrict cell wall elongation to their cell poles, but it is not known how polar growth is affected on the molecular level. Wag31 is a protein that is required for this polar elongation. In this work, we show that Wag31 actually has at least four distinct functions in regulating the cell wall: it promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at the new pole and the septum. In addition, we propose a new model for how Wag31 self-associates into a protein network. This work is important because it shows that a DivIVA homolog can have distinct functions depending on cell context. And, this work clarifies that Wag31 is doing several different things in the cell, and gives us genetic tools to disentangle its functions.

microbiology↗

bioRxiv2021

Peptidoglycan recycling contributes to outer membrane integrity and carbapenem tolerance in Acinetobacter baumannii

The Gram-negative cell envelope is an essential structure that not only protects the cell against lysis from the internal turgor, but also forms a barrier to limit entry of antibiotics. Some of our most potent bactericidal antibiotics, the {beta}-lactams, exploit the essentiality of the cell envelope by inhibiting its biosynthesis, typically inducing lysis and rapid death. However, many Gram-negative bacteria exhibit "antibiotic tolerance", the ability to sustain viability in the presence of {beta}-lactams for extended time periods. Despite several studies showing that antibiotic tolerance contributes directly to treatment failure, and is a steppingstone in acquisition of true resistance, the molecular factors that promote intrinsic tolerance are not well-understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. While typically reserved to combat multidrug resistant infections, carbapenem {beta}-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate high intrinsic tolerance to meropenem, form spheroplasts with the antibiotic and revert to normal growth after antibiotic removal. Using transcriptomics and genetics screens, we characterized novel tolerance factors and found that outer membrane integrity maintenance, drug efflux and peptidoglycan homeostasis collectively contribute to meropenem tolerance in A. baumannii. Furthermore, outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to meropenem tolerance in A. baumannii. ImportanceCarbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting an urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well-understood. Here, we characterized a role for peptidoglycan recycling in outer membrane integrity maintenance and carbapenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlights physiological processes that could be targeted to improve antimicrobial treatment.

microbiology↗

bioRxiv2020

The stringent factor Rel from Mycobacterium abscessus regulates metabolism, but does not promote survival in stress or antibiotics

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the non-tuberculous mycobacterial pathogen, Mycobacterium abscessus (Mab). We found that deletion of rel does not ablate (p)ppGpp synthesis, and that rel does not provide a survival advantage in several stress conditions, or in antibiotic treatment. Transcriptional data show that RelMab is involved in regulating expression of anabolism and growth genes in stationary phase. However, it does not activate transcription of stress response or antibiotic resistance genes, and actually represses transcription of many antibiotic resistance genes. This work shows that there is an unannotated (p)ppGpp synthetase in Mab. ImportanceIn this study, we examined the functional roles of the stringent factor Rel in Mycobacterium abscessus (Mab). In most species, stringent factors synthesize the alarmone (p)ppGpp, which globally alters transcription to promote growth arrest and survival under stress and in antibiotic treatment. Our work shows that in Mab, an emerging pathogen which is resistant to many antibiotics, the stringent factor Rel is not solely responsible for synthesizing (p)ppGpp. We find that RelMab downregulates many metabolic genes under stress, but does not upregulate stress response genes and does not promote antibiotic tolerance. This study implies that there is another critical but unannotated (p)ppGpp synthetase in Mab, and suggests that RelMab inhibitors are unlikely to sensitize Mab infections to antibiotic treatment.

microbiology↗

bioRxiv2019

Phosphorylation on PstP controls cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

microbiology↗